内毒素对人树突状细胞影响的体外研究

发布时间：2018-03-20 14:20

本文选题：树突状细胞　切入点：脂多糖　出处：《山西医科大学》2006年硕士论文　论文类型：学位论文

【摘要】： 目的本研究取正常人外周血单个核细胞(PBMC),建立体外诱导培养树突状细胞(DC)的方法,进而探讨内毒素对人DC成熟的影响。方法用rhGM-CSF、rhIL-4、Flt3-L和TNF-α体外诱导人外周血单个核细胞向DC分化,培养12天后收集DC。将细胞分为六组:①无LPS处理组;②0.5μg/ml LPS处理组;③1μg/ml LPS处理组;④1.5μg/ml LPS处理组;⑤2μg/ml LPS处理组;⑥5μg/ml LPS处理组。LPS均在培养的第11天加入。在倒置显微镜下观察细胞的形态,采用流式细胞术检测DC的表型,通过混合淋巴细胞反应检测DC刺激T淋巴细胞的能力,用ELISA法检测DC分泌细胞因子IL-12、IFN-γ的水平。结果 1、体外培养12天后获得形态典型的DC,细胞存活率均在90%以上。 2、培养第1天细胞CD14表达较高,CD80、CD83、CD86及HLA-DR表达较低,而培养12天后,CD14表达明显降低,CD80、CD83、CD86及HLA-DR表达明显升高。不同浓度LPS处理组与无LPS处理组相比,这种趋势更加明显(P0.05),但与LPS剂量依赖性不明显(P0.05)。 3、不同浓度LPS处理组刺激同种异体T细胞增殖的能力均高于无LPS处理组(P0.05),与LPS剂量依赖性不明显(P0.05)。 4、不同浓度LPS处理组分泌IL-12和IFN-γ的能力明显高于无LPS处理组(P0.05),与LPS剂量依赖性不明显(P0.05)。结论取正常人PBMC,联合应用rhGM-CSF(1000u/ml),rhIL-4(1000u/ml),rh Flt3-L(50ng/ml)及TNF-α(1000u/ml),在体外可诱导出具有典型形态、表型及生物学活性的DC。经LPS处理后DC发育更加成熟,表现为突起更加明显,特征性表面分子表达更高,刺激同种异体T淋巴细胞增殖的能力更强,分泌相关细胞因子的能力更高,在一定浓度范围内,这种特性与LPS剂量依赖性不明显。说明在DC诱导培养的后期,LPS可以进一步促进DC的成熟,由此可以进一步推断DC疫苗经体外诱导培养后注入伴有肠源性内毒素血症的肝病患者体内其疗效将会更加明显,这为DC疫苗用于内毒素血症患者的免疫治疗提供了实验依据。
[Abstract]:Objective to study the effect of endotoxin on the maturation of dendritic cells (DC) from normal human peripheral blood mononuclear cells (PBMCs). Methods Human peripheral blood mononuclear cells (PBMC) were induced to differentiate into DC by rhGM-CSF- rhIL-4Flt3-L and TNF- 伪 in vitro. After 12 days of culture, the cells were divided into six groups: 1 without LPS treatment group, 20.5 渭 g / ml LPS group, 31 渭 g / ml LPS group, 41.5 渭 g / ml LPS treatment group, 52 渭 g / ml LPS treatment group, 65 渭 g / ml LPS treatment group, respectively, on the 11th day of culture. The morphology of cells was observed under inverted microscope. The phenotype of DC was detected by flow cytometry, the ability of DC to stimulate T lymphocytes was detected by mixed lymphocyte reaction, and the level of IL-12 IFN- 纬 secreted by DC was detected by ELISA method. Results. 1. After 12 days of culture in vitro, typical DCs were obtained, and the cell survival rate was above 90%. 2. On the first day of culture, the expression of CD14 was higher than that of CD80, CD83, CD86 and HLA-DR, but the expression of CD14 decreased significantly after 12 days of culture. The expression of CD83, CD86 and HLA-DR in the cells treated with different concentrations of LPS was significantly higher than that in the group without LPS. This trend is more obvious than that of LPS, but it is not significantly related to the dose dependence of LPS. 3. The ability of stimulating the proliferation of allogeneic T cells in different concentrations of LPS treatment group was higher than that in the control group without LPS treatment (P 0.05), but not in a dose-dependent manner with LPS. 4. The ability of secreting IL-12 and IFN- 纬 in different concentration of LPS treatment group was significantly higher than that in the control group without LPS treatment, but had no significant dose-dependent relationship with LPS. Conclusion\\\;\\\. The ability of stimulating the proliferation of allogeneic T lymphocytes is stronger and the ability of secreting related cytokines is higher in a certain concentration range. This characteristic is not obvious with the dose dependence of LPS, which indicates that LPs can further promote the maturation of DC at the late stage of DC induction culture. It can be further concluded that the effect of DC vaccine injected into liver disease patients with intestinal endotoxemia after induction and culture in vitro will be more obvious, which provides experimental basis for DC vaccine to be used in immunotherapy of patients with endotoxemia.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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